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PubMed1995

Sister chromatid separation in vivo and in vitro.

The proteins that are involved in the mechanics of sister chromatid separation and that regulate this process are (as yet) largely unknown. Topoisomerase II activity is known to be essential for sister chromatid separation, probably to decatenate sister chromatids that have remained linked until the metaphase/anaphase transition, but other unidentified proteins are also required. An in vitro sister chromatid separation assay has been developed recently that complements the previously existing techniques used to study the process. This assay had led to some new insights into sister chromatid separation, showing, for example, that the process is regulated by ubiquitin-mediated proteolysis of protein(s) that are not known mitotic cyclins.

Animals↗
PubMed2007

The origin recognition complex functions in sister-chromatid cohesion in Saccharomyces cerevisiae.

High-fidelity chromosomal segregation requires the properly timed establishment of sister-chromatid cohesion mediated by the Cohesin complex, and its resolution at the metaphase-to-anaphase transition. We have examined cell-cycle progression in a yeast strain from which the origin recognition complex protein Orc2 was depleted after the assembly of prereplication complexes. We find that Orc2 depletion causes a delay in progression through mitosis, reflecting activation of both the DNA-damage and Mad2-spindle checkpoints. Surprisingly, sister-chromatid cohesion is impaired in Orc2-depleted cells, although Cohesin subunits are properly associated with chromatin. Reexpression of Orc2 in late G2/M phase restores chromatid cohesion. Finally, the targeting of Orc2 to a specific chromosomal locus suppresses premature sister-chromatid separation locally in a temperature-sensitive cohesin mutant. We conclude that ORC mediates sister-chromatid interaction on a pathway that is additive with Cohesin-mediated pairing.

Acetyltransferases↗
PubMed2005

Sororin, a substrate of the anaphase-promoting complex, is required for sister chromatid cohesion in vertebrates.

We have identified a regulator of sister chromatid cohesion in a screen for cell cycle-controlled proteins. This 35 kDa protein is degraded through anaphase-promoting complex (APC)-dependent ubiquitination in G1. The protein is nuclear in interphase cells, dispersed from the chromatin in mitosis, and interacts with the cohesin complex. In Xenopus embryos, overexpression of the protein causes failure to resolve and segregate sister chromatids in mitosis and an increase in the level of cohesin associated with metaphase chromosomes. In cultured cells, depletion of the protein causes mitotic arrest and complete failure of sister chromatid cohesion. This protein is thus an essential, cell cycle-dependent mediator of sister chromatid cohesion. Based on sequence analysis, this protein has no apparent orthologs outside of the vertebrates. We speculate that the protein, which we have named sororin, regulates the ability of the cohesin complex to mediate sister chromatid cohesion, perhaps by altering the nature of the interaction of cohesin with the chromosomes.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae.

The repair of DNA double-strand breaks by recombination requires the presence of an undamaged copy that is used as a template during the repair process. Because cells acquire resistance to gamma irradiation during DNA replication and because sister chromatids are the preferred partner for double-strand break repair in mitotic diploid yeast cells, it has long been suspected that cohesion between sister chromatids might be crucial for efficient repair. This hypothesis is consistent with the sensitivity to gamma irradiation of mutants defective in the cohesin complex that holds sister chromatids together from DNA replication until the onset of anaphase (reviewed in) . It is also in accordance with the finding that surveillance mechanisms (checkpoints) that sense DNA damage arrest cell cycle progression in yeast by causing stabilization of the securin Pds1, thereby blocking sister chromatid separation. The hypersensitivity to irradiation of cohesin mutants could, however, be due to a more direct involvement of the cohesin complex in the process of DNA repair. We show here that passage through S phase in the presence of cohesin, and not cohesin per se, is essential for efficient double-strand break repair during G2 in yeast. Proteins needed to load cohesin onto chromosomes (Scc2) and to generate cohesion during S phase (Eco1) are also shown to be required for repair. Our results confirm what has long been suspected but never proven, that cohesion between sister chromatids is essential for efficient double-strand break repair in mitotic cells.

Acetyltransferases↗
PubMed2002

Requirement of chromatid cohesion proteins rad21/scc1 and mis4/scc2 for normal spindle-kinetochore interaction in fission yeast.

BACKGROUND: Proteins conserved from yeast to human hold two sister chromatids together. The failure to form cohesion in the S phase results in premature separation of chromatids in G2/M. Mitotic kinetochores free from microtubules or the lack of tension are known to activate spindle checkpoint. RESULTS: The loss of chromatid cohesion in fission yeast mutants (mis4-242 and rad21-K1) leads to the activation of Mad2- and Bub1-dependent checkpoint, possibly due to a diminished microtubule-kinetochore interaction. Bub1, a checkpoint kinase, localizes briefly at early mitotic kinetochores in wild-type, whereas the cohesion mutation greatly increases the duration of kinetochore localization. Bub1 is bound to the central centromere region of mitotic cells. These cohesion mutants are hypersensitive to a tubulin poison and are synthetic lethal with dis1 and bir1/cut17, which are defective in microtubule-kinetochore interaction. The formation of specialized centromere chromatin containing CENP-A does not require cohesion. Dominant-negative noncleavable Rad21 fails to activate checkpoint but blocks sister chromatid separation and full spindle elongation in anaphase. CONCLUSIONS: Mis4 and Rad21 (budding yeast Scc2 and Scc1 homologs, respectively) act in establishing the normal spindle-kinetochore interaction in early mitosis and inhibit sister chromatid separation until the cleavage of Rad21 in anaphase. Checkpoint directly or indirectly monitors the states of cohesion in early mitosis. Full spindle extension occurs with unequal nuclear division in cohesion mutants in the absence of Mad2.

Cell Cycle↗
PubMed1999

Chromosomal aberrations and sister-chromatid exchanges in Lithuanian populations: effects of occupational and environmental exposures.

Cytogenetic analysis of chromosomal aberrations (CA) in 175,229 cells from 1113 individuals, both unexposed and occupationally or environmentally exposed to heavy metals (mercury and lead), organic (styrene, formaldehyde, phenol and benzo(a)pyrene) and inorganic (sulfur and nitrogen oxides, hydrogen and ammonium fluorides) volatile substances and/or ionizing radiation was performed. In addition, 11,250 cells from 225 individuals were scored for the frequency of sister-chromatid exchanges (SCE). Increased frequencies of CA were found in all occupationally exposed groups. A principal difference between the exposure to heavy metals and organic substances was found: increase in the CA frequency was dependent on duration of exposure to mercury but not dependent on duration of exposure to styrene, formaldehyde and phenol. A higher CA incidence was found in lymphocytes of children living in the vicinity of a plant manufacturing phosphate fertilizers. This indicates that children are a sensitive study group for the assessment of environmental exposure. However, the results of SCE analysis in these children were inconclusive. Exposure to ionizing radiation was found to cause chromosome breaks and chromatid exchanges in Chernobyl clean-up workers and chromatid breaks, chromatid exchanges, dicentric chromosomes and chromosome translocations in workers from the Ignalina Nuclear Power Plant. The increased frequency of chromatid exchanges in individuals exposed to ionizing radiation was quite unexpected. This may be attributed to the action of some unrecognized life-style or occupational factors, or to be a result of radiation-induced genomic instability. Also an increased SCE frequency was found in lymphocytes of Chernobyl clean-up workers.

Adolescent↗
PubMed2000

No genotoxic effect of propofol in chinese hamster ovary cells: analysis by sister chromatid exchanges.

BACKGROUND: In spite of its high placental transfer, propofol is frequently used in general anesthesia and sedation during obstetric and gynecological surgery such as in vitro fertilization. This study investigated whether or not propofol has a genotoxic potential by the sister chromatid exchange assay in vitro. METHODS: Sister chromatid exchanges induced after exposure to propofol were measured in Chinese hamster ovary cells with and without metabolic activation. After propofol (0.2-20 microg ml(-1)) diluted dimethyl sulfoxide was applied for 2 h with or without S9 mix, the cells having been incubated for two metaphases (34 h) in the presence of 5'-bromo-2-deoxyuridine. N-nitrosodimethylamine and mitomycin C were used as positive controls with and without metabolic activation. The chromosomes were stained with the fluorescence plus Giemsa method, and then sister chromatid exchanges in 50 cells were counted for each concentration. RESULTS: Although increasing concentrations of propofol inhibited cell proliferation, no concentrations of propofol used in this study increased the sister chromatid exchange values, with and without metabolic activation. CONCLUSION: It was concluded that there was no indication, from the sister chromatid exchange assay in mammalian cells, of a genotoxic effect of propofol and its metabolites.

Anesthetics, Intravenous↗
PubMed1983

Chromatid damage after G2 phase x-irradiation of cells from cancer-prone individuals implicates deficiency in DNA repair.

Ten lines of skin fibroblasts from individuals with genetic disorders predisposing to a high risk of cancer were compared with nine lines from normal adult donors with respect to chromatid damage after x-irradiation [25, 50, and 100 rad (0.25, 0.50, and 1 gray)] during G2 phase. The 10 cell lines represented five genetic disorders: Bloom syndrome, familial polyposis, Fanconi anemia, Gardner syndrome, and xeroderma pigmentosum, complementation groups A(XP-A), C(XP-C), E(XP-E), and variant (XP-Va). The incidence of chromatid breaks in all cancer-prone lines except XP-E and XP-A was significantly higher than in the normal lines. The incidence of chromatid gaps in all cancer-prone lines except XP-A and XP-Va was significantly higher than in the normal lines. Because each chromatid apparently contains a single continuous DNA double strand, chromatid breaks and gaps represent unrepaired DNA strand breaks arising directly or indirectly during excision repair of x-ray-induced DNA damage. These cytogenetic data together with results from use of the DNA repair inhibitor arabinofuranosyl cytosine (cytosine arabinoside) suggest that cells from all of these cancer-prone individuals are deficient in some step of DNA repair, predominantly excision repair operative during the G2-prophase period of the cell cycle. It appears that these DNA repair deficiencies are associated with a genetic predisposition to a high risk of cancer.

Abnormalities, Multiple↗
PubMed2000

Human chromatid cohesin component hRad21 is phosphorylated in M phase and associated with metaphase centromeres.

Sister chromatids duplicated in S phase are connected with each other during G(2) and M phase until the onset of anaphase. This chromatid cohesion is essential for correct segregation of genetic material to daughter cells. Recently, understanding of the molecular mechanisms governing chromatid cohesion in yeast has been greatly advanced, whereas these processes in mammalian cells remain unclear. We report here biochemical and cytological analyses of human Rad21, a homologue of the yeast cohesin subunit, Scc1p/Mcd1p. hRad21 is a nuclear phosphorylated protein. Its abundance does not change during the cell cycle, and it becomes hyperyphosphorylated in M phase. Most hRad21 is not associated with chromatin when the nuclear envelope breakdown takes place in prophase. However, a detailed analysis of the spread chromosomes indicated that hRad21 remains associated with prometaphase-like chromosomes along their entire lengths. The mitotic chromatin-bound hRad21 becomes dissociated in a highly regulated manner because hRad21 remains specifically at the centromeres but disappears from the arm regions on metaphase-like chromosomes. Interestingly, hRad21 at the metaphase centromeres appears to be present at the inner pairing domain where the two sister chromatids are supposed to be in intimate contact. These results suggest that hRad21 has a critical role in chromatid cohesion in human mitotic cells.

Antibodies↗
PubMed2001

Dose--response of initial G2-chromatid breaks induced in normal human fibroblasts by heavy ions.

PURPOSE: To investigate initial chromatid breaks in prematurely condensed G2 chromosomes following exposure to heavy ions of different LET. MATERIAL AND METHODS: Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (13 keV/ microm, 80 keV/microm), silicon (55 keV/microm) and iron (140 keV/microm, 185keV/microm, 440keV/microm) ions. Chromosomes were prematurely condensed using calyculin-A. Initial chromatid-type and isochromatid breaks in G2 cells were scored. RESULTS: The dose response curves for total chromatid breaks were linear regardless of radiation type. The relative biological effectiveness (RBE) showed a LET-dependent increase, peaking around 2.7 at 55-80keV/microm and decreasing at higher LET. The dose response curves for isochromatid-type breaks were linear for high-LET radiations, but linear-quadratic for gamma-rays and 13 keV/microm carbon ions. The RBE for the induction of isochromatid breaks obtained from linear components increased rapidly between 13keV/microm (about 7) and 80keV/microm carbon (about 71), and decreased gradually until 440 keV/microm iron ions (about 66). CONCLUSIONS: High-LET radiations are more effective at inducing isochromatid breaks, while low-LET radiations are more effective at inducing chromatid-type breaks. The densely ionizing track structures of heavy ions and the proximity of sister chromatids in G2 cells result in an increase in isochromatid breaks.

Azure Stains↗
PubMed1986

Mouse satellite DNA, centromere structure, and sister chromatid pairing.

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.

Animals↗
PubMed1988

Sister chromatid exchanges and chromosomal aberrations in mouse cells infected with the Abelson and Moloney leukemia viruses.

'Spontaneous' and mitomycin C (MMC)-induced sister chromatid exchanges (SCE) and chromatid breaks were scored in ANN-1 fibroblasts, a non-producer mouse cell line transformed by the Abelson murine leukemia virus (A-MuLV), a replication defective retrovirus whose genome contains the v-abl oncogene. Normal, non-transformed NIH3T3 fibroblasts were used as control. SCE and chromatid break frequencies in untreated or MMC-treated ANN-1 and NIH3T3 cells were compared with those observed in the same cells after infection with the helper murine Moloney leukemia virus (M-MuLV), which rescues the ability of A-MuLV to replicate in ANN-1 cells. The frequency of spontaneous and MMC-induced SCE were not significantly different in both ANN-1 and NIH3T3 cells, independently of M-MuLV infection. After M-MuLV infection, however, increased 'spontaneous' frequency of SCE and altered susceptibility to the induction of SCE by MMC was observed in both cell lines compared to M-MuLV-uninfected cells. In the case of chromatid breaks, the baseline frequency was not significantly different between the two cell lines both in the presence or in the absence of M-MuLV infection, nor was it significantly increased by M-MuLV, with respect to the value observed in uninfected cells. These results indicate that, at variance with what occurs with SCE, viral replication is not needed to increase the frequency of chromosomal aberrations and that the portion of A-MuLV genome alone is sufficient to increase chromatid breaks but not SCE in ANN-1 cells. Thus, in mouse cells carrying retroviruses, SCE and chromosomal aberrations seem to be independently generated, and influenced by different viral genes.

Abelson murine leukemia virus↗
PubMed1992

The Drosophila mei-S332 gene promotes sister-chromatid cohesion in meiosis following kinetochore differentiation.

The Drosophila mei-S332 gene acts to maintain sister-chromatid cohesion before anaphase II of meiosis in both males and females. By isolating and analyzing seven new alleles and a deficiency uncovering the mei-S332 gene we have demonstrated that the onset of the requirement for mei-S332 is not until late anaphase I. All of our alleles result primarily in equational (meiosis II) nondisjunction with low amounts of reductional (meiosis I) nondisjunction. Cytological analysis revealed that sister chromatids frequently separate in late anaphase I in these mutants. Since the sister chromatids remain associated until late in the first division, chromosomes segregate normally during meiosis I, and the genetic consequences of premature sister-chromatid dissociation are seen as nondisjunction in meiosis II. The late onset of mei-S332 action demonstrated by the mutations was not a consequence of residual gene function because two strong, and possibly null, alleles give predominantly equational nondisjunction both as homozygotes and in trans to a deficiency. mei-S332 is not required until after metaphase I, when the kinetochore differentiates from a single hemispherical kinetochore jointly organized by the sister chromatids into two distinct sister kinetochores. Therefore, we propose that the mei-S322 product acts to hold the doubled kinetochore together until anaphase II. All of the alleles are fully viable when in trans to a deficiency, thus mei-S332 is not essential for mitosis. Four of the alleles show an unexpected sex specificity.

Alleles↗
PubMed1992

Sister chromatids are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae.

A diploid Saccharomyces cerevisiae strain was constructed in which the products of both homolog recombination and unequal sister chromatid recombination events could be selected. This strain was synchronized in G1 or in G2, irradiated with X-rays to induce DNA damage, and monitored for levels of recombination. Cells irradiated in G1 were found to repair recombinogenic damage primarily by homolog recombination, whereas those irradiated in G2 repaired such damage preferentially by sister chromatid recombination. We found, as have others, that G1 diploids were much more sensitive to the lethal effects of X-ray damage than were G2 diploids, especially at higher doses of irradiation. The following possible explanations for this observation were tested: G2 cells have more potential templates for repair than G1 cells; G2 cells are protected by the RAD9-mediated delay in G2 following DNA damage; sister chromatids may share more homology than homologous chromosomes. All these possibilities were ruled out by appropriate tests. We propose that, due to a special relationship they share, sister chromatids are not only preferred over homologous chromatids as substrates for recombinational repair, but have the capacity to repair more DNA damage than do homologs.

DNA Damage↗
PubMed1998

Induced chromosomal exchange directs the segregation of recombinant chromatids in mitosis of Drosophila.

In meiosis, the segregation of chromosomes at the reductional division is accomplished by first linking homologs together. Genetic exchange generates the bivalents that direct regular chromosome segregation. We show that genetic exchange in mitosis also generates bivalents and that these bivalents direct mitotic chromosome segregation. After FLP-mediated homologous recombination in G2 of the cell cycle, recombinant chromatids consistently segregate away from each other (x segregation). This pattern of segregation also applies to exchange between heterologs. Most, or all, cases of non-x segregation are the result of exchange in G1. Cytological evidence is presented that confirms the existence of the bivalents that direct this pattern of segregation. Our results implicate sister chromatid cohesion in maintenance of the bivalent. The pattern of chromatid segregation can be altered by providing an additional FRT at a more proximal site on one chromosome. We propose that sister chromatid exchange occurs at the more proximal site, allowing the recombinant chromatids to segregate together. This also allowed the recovery of reciprocal translocations following FLP-mediated heterologous recombination. The observation that exchange can generate a bivalent in mitotic divisions provides support for a simple evolutionary relationship between mitosis and meiosis.

Animals↗
PubMed2001

Drosophila separase is required for sister chromatid separation and binds to PIM and THR.

Drosophila PIM and THR are required for sister chromatid separation in mitosis and associate in vivo. Neither of these two proteins shares significant sequence similarity with known proteins. However, PIM has functional similarities with securin proteins. Like securin, PIM is degraded at the metaphase-to-anaphase transition and this degradation is required for sister chromatid separation. Securin binds and inhibits separase, a conserved cysteine endoprotease. Proteolysis of securin at the metaphase-to-anaphase transition activates separase, which degrades a conserved cohesin subunit, thereby allowing sister chromatid separation. To address whether PIM regulates separase activity or functions with THR in a distinct pathway, we have characterized a Drosophila separase homolog (SSE). SSE is an unusual member of the separase family. SSE is only about one-third the size of other separases and has a diverged endoprotease domain. However, our genetic analyses show that SSE is essential and required for sister chromatid separation during mitosis. Moreover, we show that SSE associates with both PIM and THR. Although our work shows that separase is required for sister chromatid separation in higher eukaryotes, in addition, it also indicates that the regulatory proteins have diverged to a surprising degree, particularly in Drosophila.

Amino Acid Sequence↗
PubMed1991

Genetic and physical analyses of sister chromatid exchange in yeast meiosis.

We have used nonessential circular minichromosomes to monitor sister chromatid exchange during yeast meiosis. Genetic analysis shows that a 64-kb circular minichromosome undergoes sister chromatid exchange during 40% of meioses. This frequency is not reduced by the presence of a homologous linear minichromosome. Furthermore, sister chromatid exchange can be stimulated by the presence of a 12-kb ARG4 DNA fragment, which contains initiation sites for meiotic gene conversion. Using physical analysis, we have directly identified a product of sister chromatid exchange: a head-to-tail dimer form of a circular minichromosome. This dimer form is absent in a rad50S mutant strain, which is deficient in processing of the ends of meiosis-specific double-stranded breaks into single-stranded DNA tails. Our studies suggest that meiotic sister chromatid exchange is stimulated by the same mechanism as meiotic homolog exchange.

Kinetics↗
PubMed2000

Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase.

Sister chromatid cohesion is essential for cell viability. We have isolated a novel temperature-sensitive lethal mutant named eso1-H17 that displays spindle assembly checkpoint-dependent mitotic delay and abnormal chromosome segregation. At the permissive temperature, the eso1-H17 mutant shows mild sensitivity to UV irradiation and DNA-damaging chemicals. At the nonpermissive temperature, the mutant is arrested in M phase with a viability loss due to a failure to establish sister chromatid cohesion during S phase. The lethal M-phase arrest phenotype, however, is suppressed by inactivation of a spindle checkpoint. The eso1(+) gene is not essential for the onset and progression of DNA replication but has remarkable genetic interactions with those genes regulating the G(1)-S transition and DNA replication. The N-terminal two-thirds of Eso1p is highly homologous to DNA polymerase eta of budding yeast and humans, and the C-terminal one-third is homologous to budding yeast Eco1p (also called Ctf7p), which is required for the establishment of sister chromatid cohesion. Deletion analysis and determination of the mutation site reveal that the function of the Eco1p/Ctf7p-homologous domain is necessary and sufficient for sister chromatid cohesion. On the other hand, deletion of the DNA polymerase eta domain in Eso1p increases sensitivity to UV irradiation. These results indicate that Eso1p plays a dual role during DNA replication. The C-terminal region acts to establish sister chromatid cohesion, and the N-terminal region presumably catalyzes translesion DNA synthesis when template DNA contains lesions that block regular DNA replication.

Acetyltransferases↗